Elsevier

Experimental Cell Research

Volume 341, Issue 1, 1 February 2016, Pages 42-53
Experimental Cell Research

Research Article
The role of Resveratrol-induced mitophagy/autophagy in peritoneal mesothelial cells inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS

https://doi.org/10.1016/j.yexcr.2016.01.014Get rights and content

Highlights

  • Exposing HPMCs to glucose-based peritoneal dialysis solutions results in Mitochondrial ROS production.

  • Mitochondrial ROS upregulates IL-1β expression through NLRP3 inflammasome activation.

  • RSV induces mitophagy/autophagy via AMPK activation in HPMCs.

  • Inhibition of mitophagy/autophagy results in ROS generation and NLRP3 inflammasome activation.

Abstract

It has been suggested that continuous exposure of peritoneal mesothelial cells (PMCs) to high glucose-containing peritoneal dialysis (PD) solutions may result in peritoneal inflammatory injury and impairment of local peritoneal host defence. Here, we investigated the effect of glucose-based PD solutions on mitochondrial reactive oxygen species (ROS) and nod-like receptor 3 (NLRP3) inflammasome activation in human PMCs (HPMCs). Exposure of HPMCs to high glucose-based PD solutions resulted in ROS production, which can trigger NLRP3 activation, leading to IL-1β secretion. Additionally, resveratrol (RSV) treatment induced mitophagy/autophagy via adenosine monophosphate-activated protein kinase (AMPK) activation. Increased mitochondrial ROS concentrations and IL-1β upregulation were confirmed following inhibition (siRNA against Beclin1 and ATG5 or autophagy inhibitor 3MA), but not induction (RSV), of mitophagy/autophagy. Furthermore, we observed that ATG5 and Beclin1 downregulation sensitised cells to IL-1β release induced by MSU or nigericin, which is an NLRP3 inflammasome activator. RSV treatment attenuated this effect. Taken together, this study may provide a potential therapeutic strategy for peritoneal inflammatory injury via NLRP3 inflammasome activation triggered by mitochondrial ROS.

Introduction

Although peritoneal dialysis (PD) is now considered an established form of renal replacement therapy, its long-term success depends on the structural integrity of the peritoneum, an organ that did not evolve for the purpose of PD. Human peritoneal mesothelial cells (HPMCs) are a critical component of the peritoneal membrane and play a pivotal role in dialysis adequacy. Compelling evidence from in vitro and in vivo studies have highlighted the harmful nature of conventional high glucose-based PD solutions on the structural, functional, and morphologic properties of HPMCs, attributed in part to chronic intraperitoneal inflammation [1], [2].

The nod-like receptor 3 (NLRP3) inflammasome is a molecular platform activated upon signs of cellular danger to trigger innate immune defences through the maturation of pro-inflammatory cytokines, such as interleukin (IL)-1β [3]. Strong associations of a number of human heritable and acquired diseases with dysregulated inflammasome activity highlight the importance of the NLRP inflammasome in regulating immune responses [4]. One model proposed that NLRP3 is activated by a common pathway of reactive oxygen species (ROS) [5].

According to diabetes-related studies, the initial reaction of cells upon exposure to high glucose is an increased production of ROS. ROS form as by-products of oxidative phosphorylation in mitochondria. Mitochondrial ROS overproduction following hyperglycaemia has been postulated to cause redox imbalance, oxidative insults, mitochondrial dysfunction, and cell death [6], [7]. Therefore, mitochondria are speculated to be a causal link between conventional high glucose-based PD solutions and HPMC dysfunction, inflammation, and apoptosis. Maintaining a healthy population of mitochondria is thus essential for proper HPMC homoeostasis. Approaches to selective removal of mitochondria by autophagy (termed mitophagy) should be considered.

Autophagy is the process of catabolism of cellular components, such as the cytosol, organelles, and protein aggregates, through their encapsulation by a double-membrane structure known as the autophagosome [8], [9]. A well-studied type of cargo-specific autophagy is mitophagy, which is used to describe the engulfment of mitochondria into vesicles that are coated with the autophagosome marker microtubule-associated protein 1 light-chain 3 (LC3) [10]. Recent developments reveal a crucial role for the autophagy pathway and proteins in immunity and inflammation. They balance the beneficial and detrimental effects of immunity and inflammation and, thereby, may protect against infectious, autoimmune, and inflammatory diseases [11].

Resveratrol (trans-3,4′,5-trihydroxystilbene; RSV), a naturally occurring polyphenolic phytoalexin found in grapes, elicits several beneficial effects in human pathologies through its anti-oxidant, anti-inflammatory, anti-obesity, and anti-cancer properties [12], [13], [14], [15]. Interestingly, resveratrol has been reported to induce autophagy in tumour cells, neurons, renal cells, and vascular endothelial cells [16], [17], [18], [19], [20].

In this study, we investigated the effect of glucose-based PD on mitochondrial ROS production and following NLRP3 inflammasome activation in HPMCs. We also characterized the molecular mechanism of resveratrol-induced mitophagy/autophagy. In addition, the effects of mitophagy/autophagy regulation on mitochondrial ROS production and IL-1β expression through NLRP3 inflammasome activation were evaluated. This study may provide a basis for further development of a potential therapeutic strategy for protecting the peritoneum membrane in long-term PD.

Section snippets

Reagents and antibodies

Mitotracker deep red, Mitotracker green, and MitoSOX were obtained from Invitrogen. MSU (monosodium urate crystals) and nigericin were obtained from InvivoGen. Resveratrol, Rapamycin, Rotenone, Thenoyltrifluoroacetone (TTFA), Antimycin A, and 3-methyladenine (3MA) were obtained from Sigma. Anti-LC3 was purchased from Novus Biologicals. Anti-IL-1β, ATG5, Beclin1, phosphor-mammalian target of Rapamycin (mTOR), mTOR, phosphor-liver kinase B1 (LKB1), LKB1, phosphor-adenosine monophosphate-activated

The effect of glucose-based PD on mitochondrial mass and mitochondrial ROS production in HMrSV5 cells

Treatment with glucose-based PD in HMrSV5 cells resulted in increased total mitochondrial and ROS production, which were determined using three types of mitochondrial-specific labels that distinguish respiring (Mitotracker deep red), total (Mitotracker green), and ROS-generating mitochondria (MitoSOX) (Fig. 1). We also investigated the effect of blocking key enzymes of the mitochondria respiratory chain of ROS production. ROS-generating mitochondria were observed when complex I and complex III

Discussion

The introduction of PD more than three decades ago has provoked much interest in mesothelial cell biology. In the peritoneal cavity, HPMCs represent the largest population of resident cells, whose primary function is to provide a non-adhesive and protective layer against foreign particle invasion and injury to the peritoneum consequent to chemical or surgical insult [24]. Our previous study demonstrated that conventional glucose-based PD solutions downregulated the expression of Toll-like

Acknowledgement

This work was supported by the National Natural Science Foundation of China (81200555).

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